An understanding of the genetic mechanisms controlling differentiation will require that specific genes can be analyzed at the levels of transcription and translation. As a model system for neural differentiation we are analyzing the isozymes of L-glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) in the developing cerebellum of mice under in vivo and in vitro conditions. Several requirements for such an analysis have been established, these include the availability of antibodies to the individual isozymes, the identification of tissues in which the adult isozyme represents up to 0.8 percent of the total protein, and the ability to measure enzyme synthesis in vitro. In addition, genetic variants of this isozyme that effect the protein structure and enzyme protein levels are available and the transition of isozyme expression during developmet can be shown to occur in primary cell cultures and established cell lines. Our initial experimental approach will be to measure translatable mRNA by cell-free protein synthesis; however, we are attempting to purify mRNA specific for the adult isozyme so that a (3H)cDNA probe can be synthesized and used to measure mRNA molecules directly by RNA-cDNA hybridization. The procedure for purifying the mRNA will be based on immunoprecipitation of polysomes, sucrose gradient centrifugation, electrophoresis, and hybridization of (3h)cDNA, synthesized from partially purified mRNA, with mRNA isolated from a tissue that lacks the isozyme of interest. By being able to measure both translatable mRNA and mRNA molecules by hybridization we hope to address the following questions: (1) Is the increase in adult isozyme during development controlled by increases in the amount of specific mRNA? (2) Are the differences in enzyme levels in the cerebellum of inbred strains of mice controlled by differences in the amount of mRNA? (3) Is the genetic locus that controls activity levels cis-acting or trans-acting? (4) Is the reversible transition from embryonic to adult isozyme expression in ascites and solid tumors controlled at the level of transcription or translation?